The object of this proposal is to study protein-nucleotide interactions in membranes, organelles and purified enzyme systems using recently synthesized photoaffinity analogs of adenosine and guanosine nucleotides. The synthesis and utilization of the adenosine analogs have already been extensively studied in my laboratory and are currently being used by other researchers as they are now available commercially. We have also synthesized and used the guanosine derivatives. However, the synthetic procedure has not yet been optimized and the use of these derivatives as photoprobes is yet in the initial stages. Therefore, the proposed research herein will be directed toward the following problems. (1) Optimizing the synthesis and storage of the guanosine nucleotide analogs and other new analogs. (2) Investigating the use of photoaffinity analogs as "active site" directed probes in several systems which require nucleotides as substrates or regulators. (3) Specifically, my laboratory will concentrate on the ATP, GTP, cAMP and cGMP sites of adenylate and guanylate cyclase and the respective cyclic nucleotide activated protein kinases. (4) In collaboration with other laboratories we will investigate other systems such as GTP and GDP sites involved in activation of protein synthesis. (5) Photoaffinity analogs of guanosine "magic spot" compounds are being synthesized and used to elucidate the mechanism of action of these compounds. (6) 8N3ATP is being used to study the mechanism of regulation of membrane bound, ATP binding proteins, and the CaATPase.